Development of potent non-carbohydrate imidazole-based small molecule selectin inhibitors with antiinflammatory activity.

A novel series of non-carbohydrate imidazole-based selectin inhibitors has been discovered via high-throughput screening using a P-selectin ELISA-based assay system. The initial lead 1 had an IC(50) of 17 microM in the P-selectin ELISA; this potency was significantly improved via an extensive SAR exploration. One of the current lead compounds (29) has an IC(50) of 300 nM in a P-selectin ELISA; it also has good activity in P- and E-selectin cell adhesion assays and shows efficacy in vivo. These compounds represent a novel series of sLe(X) mimetics with antiinflammatory activity. Their unique profile supports our interest in their further evaluation as drug candidates for the treatment of inflammation. Herein we describe the syntheses, optimization, and SAR of this series of novel potent selectin antagonists.

High-throughput purification of compound libraries.

Synthesis of combinatorial libraries by parallel synthesis, followed by high- throughput biological screening, is the new paradigm for drug discovery. Purity of these libraries is an important consideration to obtain high-quality assay data. Liquid-liquid extraction and solid-phase capture reagents are useful in special cases for small numbers of compounds. However, for libraries of a few thousand compounds, HPLC is a viable alternative. Beyond these numbers, factors such as solvent requirements, the number of fractions and tracking become prohibitive. Supercritical fluid chromatography has been successfully employed in automated purification instrumentation and is expected to be capable of purifying libraries of tens-of-thousands of compounds.

New thrombin inhibitors in cardiovascular disease.

Thrombin is a multifunctional serine protease that plays a primary role in the pathogenic pathway of thrombosis as a consequence of its actions on the two principal components of blood clots, fibrin and activated platelets. Deficiencies in available therapies, heparin and coumadin, have recently led to an intense effort in several pharmaceutical companies to develop potent, selective, and preferably orally bioavailable direct thrombin inhibitors. Substantial progress has been made on potent and selective compounds and work is ongoing to improve other pharmacokinetic properties such as oral bioavailability and duration of action, properties necessary for successful clinical development.

Synthesis, structure, and structure-activity relationships of divalent thrombin inhibitors containing an alpha-keto-amide transition-state mimetic.

A new class of divalent thrombin inhibitors is described that contains an alpha-keto-amide transition-state mimetic linking an active site binding group and a group that binds to the fibrinogen-binding exosite. The X-ray crystallographic structure of the most potent member of this new class, CVS995, shows many features in common with other divalent thrombin inhibitors and clearly defines the transition-state-like binding of the alpha-keto-amide group. The structure of the active site part of the inhibitor shows a network of water molecules connecting both the side-chain and backbone atoms of thrombin and the inhibitor. Direct peptide analogues of the new transition-state-containing divalent thrombin inhibitors were compared using in vitro assays of thrombin inhibition. There was no direct correlation between the binding constants of the peptides and their alpha-keto-amide counterparts. The most potent alpha-keto-amide inhibitor, CVS995, with a Ki = 1 pM, did not correspond to the most potent divalent peptide and contained a single amino acid deletion in the exosite binding region with respect to the equivalent region of the natural thrombin inhibitor hirudin. The interaction energies of the active site, transition state, and exosite binding regions of these new divalent thrombin inhibitors are not additive.

Characterisation of a novel series of aprotinin-derived anticoagulants. I. In vitro and pharmacological properties.

Previous investigations have indicated that interference with the initial level of the blood coagulation may lead to effective antithrombotic therapy. Recently a series of potential coagulation inhibitors derived from bovine pancreatic trypsin inhibitor (BPTI, aprotinin) was described. We have determined their inhibition constants, effects on coagulation assays, effects in an in vitro human thrombosis model and pharmacological profiles in hamsters. The aprotinin-derived analogues (4C2, 7L22, 5L15, 6L15, 5L84) showed significantly increased inhibitory activity towards factor Xa, factor VIIa-tissue factor (TF) complex, factor XIa and plasma kallikrein or a combination of them, and a significantly decreased plasmin inhibition as compared to aprotinin. In the coagulation assays, 4C2 and 7L22 mainly inhibited factor Xa, 5L15 and 6L15 inhibited factor VIIa-TF complex and 5L84 inhibited factor Xa, factor VIIa-TF complex and the contact activation. In flow chamber experiments with human blood 7L22, 5L15, 6L15, 5L84 and rTAP significantly inhibited fibrin formation and platelet deposition on extracellular matrix from phorbol ester stimulated human endothelial cells both under high and low shear stress and in the presence of low molecular weight heparin. The pharmacological profiles of the aprotinin analogues and rTAP with a mean residence time of 64 to 140 min were not significantly different. Modification of an aprotinin analogue with PEG (5L15-PEG) resulted in a 10-fold decrease of the inhibition constant for the factor VIIa-TF complex and in a significant prolongation of the secondary half-life, while the initial half-life was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Modeling rhodopsin, a member of G-protein coupled receptors, by computer graphics. Interpretation of chemical shifts of fluorinated rhodopsins.

An attempt has been made to construct a 3-D model of rhodopsin, a member of G-protein coupled receptors. Sequence homology of rhodopsin with the latter was a factor considered in the modeling procedure. The constructed model has been used to compare currently available specific protein/substrate interaction information, the shape of the binding cavity derived from shape of binding retinal isomers and analogs and challenged to explain recently available results from a series of fluorinated rhodopsins.

Synthesis and NMR conformational analysis of a beta-turn mimic incorporated into gramicidin S. A general approach to evaluate beta-turn peptidomimetics.

The solution structure of a gramicidin S (GS) analog containing a beta-turn mimic [BTD4-5, Lys2.2']GS has been compared to that of native GS. The linear [BTD4-5, Lys2.2']GS was synthesized by solid phase methodology and the cyclized peptide was analyzed by NMR. In the peptide portion of [BTD4-5, Lys2.2']GS, the intramolecular hydrogen bonding pattern, inter-residue NOEs, including a transannular H alpha-H alpha NOE, and JN alpha coupling constants all describe a solution structure which is equivalent to that of native GS. These data confirm that the BTD group is a competent Type II' beta-turn mimic since it does not disrupt the native conformation of GS. It also supports the use of GS as a conformational model in which to test beta-turn mimics.

Computer modelling of the NAD binding site of ADP-ribosylating toxins: active-site structure and mechanism of NAD binding.

Five ADP-ribosylating bacterial toxins, pertussis toxin, cholera toxin, diphtheria toxin, Escherichia LT toxin and Pseudomonas exotoxin A, show significant homology in selected segments of their sequence. Site-directed mutagenesis and chemical modification of residues within these regions cause loss of catalytic activity and of NAD binding. On the basis of these results and of molecular modelling based on the three-dimensional structure of exotoxin A, the geometry of an NAD binding site common to all the toxins is deduced and described in the paper. For diphtheria toxin, sequence similarity with exotoxin A is such that its preliminary structure can be computed by molecular modelling, whereas for the other toxins similarity appears to be restricted to the NAD binding site. Moreover, an analysis of molecular fitting of the NAD molecule into its binding cavity suggests a new model for the conformation of the bound NAD that better accounts for all available experimental information.

Molecular modeling in the design of phospholipase A2 inhibitors.

The X-ray structures of pancreatic bovine and porcine phospholipases A2 have been used along with interactive computer graphics to design conformationally rigid, novel compounds (1-meta-hydroxybenzyl-2-substituted acenaphthenes) directed at the active sites of these enzymes. In vitro testing confirmed that the designed compounds are potent inhibitors of the porcine pancreatic phospholipase A2 and exhibit both stereoselectivity and structure-activity relationships that are consistent with the proposed mode of binding. These compounds take advantage of a hydrophobic "slot" positioned between residues Leu-2 and Tyr-69 while positioning hydrogen-bonding functionality directed at the nd1-N of His-48. Experimental evidence shows a regioselective preference for this H-bond acceptor. A second part of the strategy used a tethered amine to displace the essential calcium providing a bisubstrate analog.

Study of the shape of the binding site of bovine opsin using 10-substituted retinal isomers.

The 9-cis, 11-cis, 13-cis, and all-trans isomers of 10-fluoro-, 10-chloro-, 10-methyl-, and 10-ethylretinals have been prepared and characterized. Results of their interaction with bovine opsin are reported. The data have been analyzed in terms of the conformational properties of the retinal isomers and their steric compatibility with the binding site as defined by the two-dimensional map disclosed earlier. The need to expand the active zone and previously undetected restrictions in the third dimension are noted.

Introduction of restriction enzyme sites in protein-coding DNA sequences by site-specific mutagenesis not affecting the amino acid sequence: a computer program.

Structure/function relationship studies of proteins are greatly facilitated by recombinant DNA technology which allows specific amino acid mutations to be made at the DNA sequence level by site-specific mutagenesis employing synthetic oligonucleotides. This technique has been successfully used to alter one or two amino acids in a protein. Replacement of existing DNA sequence coding for several amino acids with new synthetic DNA fragments would be facilitated by the presence of unique restriction enzyme sites in the region of interest. This computer program provides a means of searching the DNA sequence of interest for restriction enzyme sites that could be introduced by site-specific mutagenesis not affecting the amino acid sequence of the protein. Alternately, the program will also allow single amino acid changes to be made.